The cellular src gene product has served as a model protein for studies on the regulation of the functional activity of a tyrosine protein kinase and for investigations of the mechanism of oncogenic activation of this class of proto-oncogene products. One project in this proposal is designed to further delineate, and characterize, the regions within the amino-terminal half of the c- src protein that quantitatively and qualitatively regulate its functional activity. The analysis of mutant forms of the c-src gene product that contain mutations within the amino-terminal half of pp60c-src suggest at least two distinct regulatory regions in this half of the molecule: alterations in one region cause a partial activation of transforming activity and an enhancement in intracellular tyrosine phosphorylation; alterations in a second region cause dramatic changes in the phenotype of transforming variants of pp60c-src, possibly by altering substrate selection in vivo. We will construct a series of new mutants to further characterize the borders of these regulatory regions and the nature of the regulation mediated within this domain of pp60c- src. We will also examine phosphorylation of tyrosine 527, which has been proposed to have a negative effect of the tyrosine kinase activity and the oncogenicity of pp60c-src. The experiments described in this proposal will examine the conditions that affect phosphorylation of tyrosine 527, and examine whether this event is mediated by autophosphorylation, or by another tyrosine kinase. These studies will involve mutational alterations which change the localization of pp60c-src and which prevent intramolecular autophosphorylation. In addition, we will analyse the expression of pp60c-src in yeast, where phosphorylation by endogenous tryosine kinases does not take place, and will examine the conditions that affect the phosphorylation of pp60c-src on tyrosine 527 in vitro in animal cells to identify another putative tryosine 527 kinase, or to determine the conditions that are necessary for autophosphorylation at this site). Finally, we will continue to investigate the interaction of pp60c- src with the polyoma virus middle T antigen in order to determine the regions of pp60c-src that are necessary for the interaction with middle T antigen and for activation of pp60c-src middle T antigen. This will serve as a model system for studying how protein-protein interactions can regulate the activity of pp60c- src.